Ionizable cationic lipid for RNA delivery

ABSTRACT

What is described is a compound of formula I 
                         
wherein
         R is a linear alkyl of 1 to 12 carbons, or a linear alkenyl or alkynyl of 2 to 12 carbons;   L is a linear alkylene or alkenylene of 5 to 18 carbons;   X is —CO—O— or —O—CO—;   Y is S or O;   R 1  is a linear or branched alkylene consisting of 1 to 6 carbons; and   R 2  and R 3  are the same or different, consisting of a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; and   n is 1-6;
 
or a salt, a solvate, or a pharmaceutical formulation thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application a divisional of U.S. patent application Ser. No.14/707,796, filed May 8, 2015, which is a continuation-in-part andclaims benefit under 35 U.S.C. §120 to U.S. patent application Ser. No.14/546,105 filed on Nov. 18, 2014, which claims benefit under 35 U.S.C.§119(e) of Provisional U.S. Patent Application No. 61/905,724, filedNov. 18, 2013, the contents of which are incorporated herein byreference in its entirety.

The description of U.S. patent application Ser. No. 14/707,876, entitled“IONIZABLE CATIONIC LIPID FOR RNA DELIVERY,” filed May 8, 2015, now U.S.Pat. No. 9,365,610, issued Jun. 14, 2016, is hereby incorporated byreference in its entirety.

BACKGROUND

A number of different types of nucleic acids are currently beingdeveloped as therapeutics for the treatment of a number of diseases.These nucleic acids include DNA in gene therapy, plasmids-basedinterfering nucleic acids, small interfering nucleic acids for use inRNA interference (RNAi), including siRNA, miRNA, antisense molecules,ribozymes and aptamers. As these molecules are being developed, therehas been developed a need to produce them in a form that is stable andhas a long shelf-life and that can be easily incorporated into ananhydrous organic or anhydrous polar aprotic solvent to enableencapsulations of the nucleic acids without the side-reactions that canoccur in a polar aqueous solution or nonpolar solvents.

The present invention relates to novel lipid compositions thatfacilitate the intracellular delivery of biologically active andtherapeutic molecules. The present invention relates also topharmaceutical compositions that comprise such lipid compositions, andthat are useful to deliver therapeutically effective amounts ofbiologically active molecules into the cells of patients.

The delivery of a therapeutic compound to a subject is important for itstherapeutic effects and usually it can be impeded by limited ability ofthe compound to reach targeted cells and tissues. Improvement of suchcompounds to enter the targeted cells of tissues by a variety of meansof delivery is crucial. The present invention relates the novel lipids,in compositions and methods for preparation that facilitate the targetedintracellular delivery of biological active molecules.

Examples of biologically active molecules for which effective targetingto a patient's tissues is often not achieved include: (1) numerousproteins including immunoglobin proteins, (2) polynucleotides such asgenomic DNA, cDNA, or mRNA (3) antisense polynucleotides; and (4) manylow molecular weight compounds, whether synthetic or naturallyoccurring, such as the peptide hormones and antibiotics.

One of the fundamental challenges now facing medical practitioners isthat a number of different types of nucleic acids are currently beingdeveloped as therapeutics for the treatment of a number of diseases.These nucleic acids include DNA in gene therapy, plasmids, smallinterfering nucleic acids (siNA), siRNA, and microRNA (miRNA) for use inRNA interference (RNAi), antisense molecules, ribozymes, antagomirs, andaptamers. As these nucleic acids are being developed, there is a need toproduce lipid formulations that are easy to make and can be readilydelivered to a target tissue.

SUMMARY

What is described herein is compounds of formula I, II, III, and IV.

What is described is a compound of formula I

in whichR₁ and R₂ both consist of a linear alkyl consisting of 1 to 9 carbons,an alkenyl or alkynyl consisting of 2 to 11 carbons;L₁ and L₂ both consist of a linear alkylene or alkenylene consisting of5 to 18 carbons, or forming a heterocycle with N;X₁ and X₃ both consist of —CO—O—;X₂ consists of S or O;L₃ consists of bond or a linear alkylene consisting of 1 to 6 carbons,or forming a heterocycle with N;R₃ consists of a linear or branched alkylene consisting of 1 to 6carbons; andR₄ and R₅ are the same or different, each consisting of a hydrogen or alinear or branched alkyl consisting of 1 to 6 carbons,or a pharmaceutically acceptable salt thereof.

What is also described herein is a compound of Formula II

in whichR₁ and R₂ both consist of a linear alkyl consisting of 1 to 12 carbons,an alkenyl or alkynyl consisting of 2 to 12 carbons,L₁ and L₂ both consist of a linear alkylene or alkenylene consisting of5 to 18 carbons, or forming a heterocycle with N,X is S,L₃ is a bond or a linear alkylene consisting of 1 to 6 carbons, orforming a heterocycle with N,R₃ is a linear or branched alkylene consisting of 1 to 6 carbons, andR₄ and R₅ are the same or different, each a hydrogen or a linear orbranched alkyl consisting of 1 to 6 carbons;or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound of formula II, L₁ and L₂ both consistof a linear alkylene consisting of five carbons. In another embodimentof the compound of formula I, R₃ is ethylene or propylene. In anotherembodiment of the compound of formula I, R₄ and R₅ are the same ordifferent, each hydrogen, methyl, or ethyl. In another embodiment of thecompound of formula I, L₃ is a bond. In another embodiment of thecompound of formula I, R₁ and R₂ both consist of a linear alkenylconsisting of ten carbons.

What is also described herein is a compound of formula III or IV

whereinR₁ is a branched alkyl with 12 to 20 carbons,R₂ is a linear alkyl with 5 to 10 carbons or a branched alkyl with 12 to20 carbons,L₁ and L₂ each consist of a bond or a linear alkyl having 1 to 3 carbonatoms,X is S or O,L₃ consists of a bond or a lower alkyl,R₃ is a lower alkyl, andR₄ and R₅ are the same or different, each a lower alkyl;or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound of formula III or IV, L₃ is consistsof a bond. Another embodiment of the compound of formula III or IV, XisS. In another embodiment of the compound of formula III or IV, R₃ isethylene. In another embodiment of the compound of formula III or IV, R₃is n-propylene or isopropylene. In another embodiment of the compound offormula III or IV, R₄ and R₅ are separately methyl, ethyl, or isopropyl.In another embodiment of the compound of formula III or IV, L₁ and L₂each consist of a bond. In another embodiment of the compound of formulaIII or IV, L₁ and L₂ each consist of a methylene. In another embodimentof the compound of formula III or IV, R₁ and R₂ each consist of branchedalkyl. In another embodiment of the compound of formula III or IV, R₂consists of an alkyl. In another embodiment of the compound of formulaIII or IV, R₁ and R₂ each consists of 19 or 20 carbon atoms. In anotherembodiment of the compound of formula III or IV, R₁ or R₂ each consistsof 13 or 14 carbon atoms. In another embodiment of the compound offormula III or IV, L₃ consists of methylene, R₃ is ethylene, X₂ is S,and R₄ and R₅ are each methyl. In another embodiment of the compound offormula III or IV, wherein L₃ consists of a bond, R₃ is ethylene, X isS, and R₄ and R₅ are each methyl. In another embodiment of the compoundof formula III or IV, L₃ consists of a bond, R₃ consists of n-propylene,X consists of S, and R₄ and R₅ each consist of methyl. In anotherembodiment of the compound of formula III or IV, L₃ consists of a bond,R₃ consists of isopropylene, X consists of S, and R₄ and R₅ each consistof methyl.

The nucleic acid preferably has an activity of suppressing theexpression of a target gene. The target gene preferably is a geneassociated with inflammation.

What is also described herein is a method for introducing a nucleic acidinto a cell of a mammal by using any of the compositions, above. Thecell may be in a liver, lung, kidney, brain, blood, spleen, or bone. Thecomposition preferably is administered intravenously, subcutaneously,intraperitoneally, or intrathecally. Preferably, the compositionsdescribed herein are used in a method for treating cancer orinflammatory disease. The disease may be one selected from the groupconsisting of immune disorder, cancer, renal disease, fibrotic disease,genetic abnormality, inflammation, and cardiovascular disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the knockdown activity of siRNA encapsulated by differentcationic lipids. The lipids include MC3 (0.3 mg/kg), NC1 (0.3 mg/kg),ATX-547 (0.3 mg/kg), ATX-001 (0.3 and 1.0 mg/kg), ATX-002 (0.3 and 1.0mg/kg), and ATX-003 (0.3 and 1.0 mg/kg). The amount of Factor VIIknockdown in mouse plasma is shown following administration of the siRNAformulation to C57BL6 mice, compared to injection of vehicle alone. Theamount of Factor VII in abnormal and normal human plasma is included asa control. Statistically significant decreases in Factor VII levels(p<0.01) is shown by an asterisk (*).

FIG. 2 shows an evaluation of the effect of siRNA of Factor VII activitybased on the results shown in FIG. 2, and normalized to percentageknockdown compared to the vehicle alone.

FIG. 3 shows the knockdown activity of siRNA encapsulated by differentcationic lipids. The lipids include MC3 (0.3 and 1.5 mg/kg), NC1 (0.3mg/kg), AT547 (0.1 and 0.3 mg/kg), AT004 (0.3), AT006 (0.3 and 1.0mg/kg), ATX-010 (0.3 mg/kg), and AT001 (0.3 and 1.5 mg/kg). The amountof Factor VII knockdown in mouse plasma is shown followingadministration of the siRNA formulation to C57BL6 mice, compared toinjection of vehicle alone. The amount of Factor VII in abnormal andnormal human plasma is included as a control. Statistically significantdecreases in Factor VII levels (p<0.01) is shown by an asterisk (*).

FIG. 4 shows an evaluation of the effect of siRNA of Factor VII activitybased on the results shown in FIG. 2, and normalized to percentageknockdown compared to the vehicle alone.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

“At least one” means one or more (e.g., 1-3, 1-2, or 1).

“Composition” includes a product comprising the specified ingredients inthe specified amounts, as well as any product that results, directly orindirectly, from combination of the specified ingredients in thespecified amounts.

“In combination with” as used to describe the administration of acompound of formulas 1, I, and II with other medicaments in the methodsof treatment of this invention, means—that the compounds of formulas 1,I, and II and the other medicaments are administered sequentially orconcurrently in separate dosage forms, or are administered concurrentlyin the same dosage form.

“Mammal” means a human or other mammal, or means a human being.

“Patient” includes both human and other mammals, preferably human.

“Alkyl” is a saturated or unsaturated, straight or branched, hydrocarbonchain. In various embodiments, the alkyl group has 1-18 carbons, i.e. isa C₁-C₁₈ group, or is a C₁-C₁₂ group, a C₁-C₆ group, or a C₁-C₄ group.Independently, in various embodiments, the alkyl group has zero branches(i.e., is a straight chain), one branch, two branches, or more than twobranches. “Alkenyl” is an unsaturated alkyl that may have one doublebond, two double bonds, or more than two double bonds. “Alkynal” is anunsaturated alkyl that may have one triple bond, two triple bonds, ormore than two triple bonds. Alkyl chains may be optionally substitutedwith 1 substituent (i.e., the alkyl group is mono-substituted), or 1-2substituents, or 1-3 substituents, or 1-4 substituents, etc. Thesubstituents may be selected from the group consisting of hydroxy,amino, alkylamino, boronyl, carboxy, nitro, cyano, and the like. Whenthe alkyl group incorporates one or more heteroatoms, the alkyl group isreferred to herein as a heteroalkyl group. When the substituents on analkyl group are hydrocarbons, then the resulting group is simplyreferred to as a substituted alkyl. In various aspects, the alkyl groupincluding substituents has less than 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 carbons.

“Lower alkyl” means a group having one to six carbons in the chain whichchain may be straight or branched. Non-limiting examples of suitablealkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, n-pentyl, and hexyl.

“Alkoxy” means an alkyl-O-group wherein alkyl is as defined above.Non-limiting examples of alkoxy groups include: methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy and heptoxy. The bond to the parentmoiety is through the ether oxygen.

“Alkoxyalkyl” means an alkoxy-alkyl-group in which the alkoxy and alkylare as previously described. Preferred alkoxyalkyl comprise a loweralkyl group. The bond to the parent moiety is through the alkyl.

“Alkylaryl” means an alkyl-aryl-group in which the alkyl and aryl are aspreviously described. Preferred alkylaryls comprise a lower alkyl group.The bond to the parent moiety is through the aryl.

“Aminoalkyl” means an NH2-alkyl-group, wherein alkyl is as definedabove, bound to the parent moiety through the alkyl group.

“Carboxyalkyl” means an HOOC-alkyl-group, wherein alkyl is as definedabove, bound to the parent moiety through the alkyl group.

“Commercially available chemicals” and the chemicals used in theExamples set forth herein may be obtained from standard commercialsources, where such sources include, for example, Acros Organics(Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), AvocadoResearch (Lancashire, U.K.), Bionet (Cornwall, U.K.), Boron Molecular(Research Triangle Park, N.C.), Combi-Blocks (San Diego, Calif.),Eastman Organic Chemicals, Eastman Kodak Company (Rochester, N.Y.),Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan,Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis(Windham, N.H.), Maybridge Chemical Co. (Cornwall, U.K.), PierceChemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany),Spectrum Quality Product, Inc. (New Brunswick, N.J.), TCI America(Portland, Oreg.), and Wako Chemicals USA, Inc. (Richmond, Va.).

“Compounds described in the chemical literature” may be identifiedthrough reference books and databases directed to chemical compounds andchemical reactions, as known to one of ordinary skill in the art.Suitable reference books and treatise that detail the synthesis ofreactants useful in the preparation of compounds disclosed herein, orprovide references to articles that describe the preparation ofcompounds disclosed herein, include for example, “Synthetic OrganicChemistry”, John Wiley and Sons, Inc. New York; S. R. Sandler et al,“Organic Functional Group Preparations,” 2nd Ed., Academic Press, NewYork, 1983; H. O. House, “Modern Synthetic Reactions,” 2nd Ed., W. A.Benjamin, Inc. Menlo Park, Calif., 1972; T. L. Glichrist, “HeterocyclicChemistry,” 2nd Ed. John Wiley and Sons, New York, 1992; J. March,“Advanced Organic Chemistry: reactions, Mechanisms and Structure,” 5thEd., Wiley Interscience, New York, 2001; Specific and analogousreactants may also be identified through the indices of known chemicalsprepared by the Chemical Abstract Service of the American ChemicalSociety, which are available in most public and university libraries, aswell as through online databases (the American Chemical Society,Washington, D.C. may be contacted for more details). Chemicals that areknown but not commercially available in catalogs may be prepared bycustom chemical synthesis houses, where many of the standard chemicalsupply houses (such as those listed above) provide custom synthesisservices.

“Halo” means fluoro, chloro, bromo, or iodo groups. Preferred arefluoro, chloro or bromo, and more preferred are fluoro and chloro.

“Halogen” means fluorine, chlorine, bromine, or iodine. Preferred arefluorine, chlorine and bromine.

“Heteroalkyl” is a saturated or unsaturated, straight or branched, chaincontaining carbon and at least one heteroatom. The heteroalkyl groupmay, in various embodiments, have on heteroatom, or 1-2 heteroatoms, or1-3 heteroatoms, or 1-4 heteroatoms. In one aspect the heteroalkyl chaincontains from 1 to 18 (i.e., 1-18) member atoms (carbon andheteroatoms), and in various embodiments contain 1-12, or 1-6, or 1-4member atoms. Independently, in various embodiments, the heteroalkylgroup has zero branches (i.e., is a straight chain), one branch, twobranches, or more than two branches. Independently, in one embodiment,the heteroalkyl group is saturated. In another embodiment, theheteroalkyl group is unsaturated. In various embodiments, theunsaturated heteroalkyl may have one double bond, two double bonds, morethan two double bonds, and/or one triple bond, two triple bonds, or morethan two triple bonds. Heteroalkyl chains may be substituted orunsubstituted. In one embodiment, the heteroalkyl chain isunsubstituted. In another embodiment, the heteroalkyl chain issubstituted. A substituted heteroalkyl chain may have 1 substituent(i.e., by monosubstituted), or may have, e.g., 1-2 substituents, or 1-3substituents, or 1-4 substituents. Exemplary heteroalkyl substituentsinclude esters (—C(O)—O—R) and carbonyls (—C(O)—).

“Hydroxyalkyl” means an HO-alkyl-group, in which alkyl is previouslydefined. Preferred hydroxyalkyls contain lower alkyl. Non-limitingexamples of suitable hydroxyalkyl groups include hydroxymethyl and2-hydroxyethyl.

“Hydrate” is a solvate wherein the solvent molecule is H₂O.

“Lipid” means an organic compound that comprises an ester of fatty acidand is characterized by being insoluble in water, but soluble in manyorganic solvents. Lipids are usually divided into at least threeclasses: (1) “simple lipids,” which include fats and oils as well aswaxes; (2) “compound lipids,” which include phospholipids andglycolipids; and (3) “derived lipids” such as steroids.

“Lipid particle” means a lipid formulation that can be used to deliver atherapeutic nucleic acid (e.g., mRNA) to a target site of interest(e.g., cell, tissue, organ, and the like). In preferred embodiments, thelipid particle is a nucleic acid-lipid particle, which is typicallyformed from a cationic lipid, a non-cationic lipid (e.g., aphospholipid), a conjugated lipid that prevents aggregation of theparticle (e.g., a PEG-lipid), and optionally cholesterol. Typically, thetherapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipidportion of the particle, thereby protecting it from enzymaticdegradation.

Lipid particles typically have a mean diameter of from 30 nm to 150 nm,from 40 nm to 150 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from70 nm to 110 nm, from 70 nm to 100 nm, from 80 nm to 100 nm, from 90 nmto 100 nm, from 70 to 90 nm, from 80 nm to 90 nm, from 70 nm to 80 nm,or 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm,80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantiallynon-toxic. In addition, nucleic acids, when present in the lipidparticles of the present invention, are resistant in aqueous solution todegradation with a nuclease.

“Solvate” means a physical association of a compound of this disclosurewith one or more solvent molecules. This physical association involvesvarying degrees of ionic and covalent bonding, including hydrogenbonding. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates, and the like.

“Lipid encapsulated” can mean a lipid particle that provides atherapeutic nucleic acid such as an mRNA with full encapsulation,partial encapsulation, or both. In a preferred embodiment, the nucleicacid (e.g., mRNA) is fully encapsulated in the lipid particle.

“Lipid conjugate” means a conjugated lipid that inhibits aggregation oflipid particles. Such lipid conjugates include, but are not limited to,PEG-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls(e.g., PEG-DAA conjugates), PEG coupled to diacylglycerols (e.g.,PEG-DAG conjugates), PEG coupled to cholesterol, PEG coupled tophosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEGlipids, polyoxazoline (POZ)-lipid conjugates, polyamide oligomers, andmixtures thereof. PEG or POZ can be conjugated directly to the lipid ormay be linked to the lipid via a linker moiety. Any linker moietysuitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester-containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester-containing linker moieties, such as amides or carbamates, areused.

“Amphipathic lipid” means the material in which the hydrophobic portionof the lipid material orients into a hydrophobic phase, while thehydrophilic portion orients toward the aqueous phase. Hydrophiliccharacteristics derive from the presence of polar or charged groups suchas carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl,nitro, hydroxyl, and other like groups. Hydrophobicity can be conferredby the inclusion of apolar groups that include, but are not limited to,long-chain saturated and unsaturated aliphatic hydrocarbon groups andsuch groups substituted by one or more aromatic, cycloaliphatic, orheterocyclic group(s). Examples of amphipathic compounds include, butare not limited to, phospholipids, aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

“Neutral lipid” means a lipid species that exist either in an unchargedor neutral zwitterionic form at a selected pH. At physiological pH, suchlipids include, for example, diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin,cholesterol, cerebrosides, and diacylglycerols.

“Non-cationic lipid” means an amphipathic lipid or a neutral lipid oranionic lipid, and is described in more detail below.

“Anionic lipid” means a lipid that is negatively charged atphysiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids of the invention may also betermed titratable cationic lipids. In some embodiments, the cationiclipids comprise: a protonatable tertiary amine (e.g., pH-titratable)head group; C₁₈ alkyl chains, wherein each alkyl chain independently has0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and ether, ester, or ketallinkages between the head group and alkyl chains. Such cationic lipidsinclude, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA,γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2,and C2K), DLin-K-C3-DM A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA,DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3) and(DLin-MP-DMA)(also known as 1-Bl 1).

The term “substituted” means substitution with specified groups otherthan hydrogen, or with one or more groups, moieties, or radicals whichcan be the same or different, with each, for example, beingindependently selected.

By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acidmolecule that binds to target RNA by means of RNA-RNA or RNA-DNA orRNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566)interactions and alters the activity of the target RNA (for a review,see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.No. 5,849,902). Typically, antisense molecules are complementary to atarget sequence along a single contiguous sequence of the antisensemolecule. However, in certain embodiments, an antisense molecule canbind to substrate such that the substrate molecule forms a loop, and/oran antisense molecule can bind such that the antisense molecule forms aloop. Thus, the antisense molecule can be complementary to two (or evenmore) non-contiguous substrate sequences or two (or even more)non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. In addition, antisense DNAcan be used to target RNA by means of DNA-RNA interactions, therebyactivating RNase H, which digests the target RNA in the duplex. Theantisense oligonucleotides can comprise one or more RNAse H activatingregion, which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof “AntisenseRNA” is an RNA strand having a sequence complementary to a target genemRNA, that can induce RNAi by binding to the target gene mRNA.“Antisense RNA” is an RNA strand having a sequence complementary to atarget gene mRNA, and thought to induce RNAi by binding to the targetgene mRNA. “Sense RNA” has a sequence complementary to the antisenseRNA, and annealed to its complementary antisense RNA to form iNA. Theseantisense and sense RNAs have been conventionally synthesized with anRNA synthesizer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2′-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution, and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of an interfering RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA. As used herein, the terms “ribonucleic acid”and “RNA” refer to a molecule containing at least one ribonucleotideresidue, including siRNA, antisense RNA, single stranded RNA, microRNA,mRNA, noncoding RNA, and multivalent RNA. A ribonucleotide is anucleotide with a hydroxyl group at the 2′ position of aβ-D-ribo-furanose moiety. These terms include double-stranded RNA,single-stranded RNA, isolated RNA such as partially purified RNA,essentially pure RNA, synthetic RNA, recombinantly produced RNA, as wellas modified and altered RNA that differs from naturally occurring RNA bythe addition, deletion, substitution, modification, and/or alteration ofone or more nucleotides. Alterations of an RNA can include addition ofnon-nucleotide material, such as to the end(s) of an interfering RNA orinternally, for example at one or more nucleotides of an RNA nucleotidesin an RNA molecule include non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar, and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate, and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein, et al., International PCT Publication No. WO92/07065; Usman, et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183,1994. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include: inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g., 6-methyluridine), propyne, and others(Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine, and uracil at 1′ position or theirequivalents.

As used herein complementary nucleotide bases are a pair of nucleotidebases that form hydrogen bonds with each other. Adenine (A) pairs withthymine (T) or with uracil (U) in RNA, and guanine (G) pairs withcytosine (C). Complementary segments or strands of nucleic acid thathybridize (i.e. join by hydrogen bonding) with each other. By“complementary” is meant that a nucleic acid can form hydrogen bond(s)with another nucleic acid sequence either by traditional Watson-Crick orby other non-traditional modes of binding.

MicroRNAs (miRNA) are single-stranded RNA molecules of 21-23 nucleotidesin length, which regulate gene expression miRNAs are encoded by genesthat are transcribed from DNA but not translated into protein(non-coding RNA); instead they are processed from primary transcriptsknown as pri-miRNA to short stem-loop structures called pre-miRNA andfinally to functional miRNA. Mature miRNA molecules are partiallycomplementary to one or more messenger RNA (mRNA) molecules, and theirmain function is to downregulate gene expression

As used herein the term “small interfering RNA (siRNA)”, sometimes knownas short interfering RNA or silencing RNA, is used to refer to a classof double-stranded RNA molecules, 16-40 nucleotides in length, that playa variety of roles in biology. Most notably, siRNA is involved in theRNA interference (RNAi) pathway, where it interferes with the expressionof a specific gene. In addition to their role in the RNAi pathway,siRNAs also act in RNAi-related pathways, e.g., as an antiviralmechanism or in shaping the chromatin structure of a genome; thecomplexity of these pathways is only now being elucidated.

As used herein, the term RNAi refers to an RNA-dependent gene silencingprocess that is controlled by the RNA-induced silencing complex (RISC)and is initiated by short double-stranded RNA molecules in a cell, wherethey interact with the catalytic RISC component argonaute. When thedouble-stranded RNA or RNA-like iNA or siRNA is exogenous (coming frominfection by a virus with an RNA genome or from transfected iNA orsiRNA), the RNA or iNA is imported directly into the cytoplasm andcleaved to short fragments by the enzyme dicer. The initiating dsRNA canalso be endogenous (originating in the cell), as in pre-microRNAsexpressed from RNA-coding genes in the genome. The primary transcriptsfrom such genes are first processed to form the characteristic stem-loopstructure of pre-miRNA in the nucleus, then exported to the cytoplasm tobe cleaved by dicer. Thus, the two dsRNA pathways, exogenous andendogenous, converge at the RISC complex. The active components of anRNA-induced silencing complex (RISC) are endonucleases called argonauteproteins, which cleave the target mRNA strand complementary to theirbound siRNA or iNA. As the fragments produced by dicer aredouble-stranded, they could each in theory produce a functional siRNA oriNA. However, only one of the two strands, which is known as the guidestrand, binds the argonaute protein and directs gene silencing. Theother anti-guide strand or passenger strand is degraded during RISCactivation.

What are described herein are compounds of formula I, II, III, and IV.

in whichR₁ and R₂ both consist of a linear alkyl consisting of 1 to 9 carbons,an alkenyl or alkynyl consisting of 2 to 11 carbons;L₁ and L₂ both consist of a linear alkylene or alkenylene consisting of5 to 18 carbons, or forming a heterocycle with N;X₁ and X₃ both consist of —CO—O—;X₂ is S or O;L₃ consists of a bond or a linear alkylene consisting of 1 to 6 carbons,or forming a heterocycle with N;R₃ consists of a linear or branched alkylene consisting of 1 to 6carbons; andR₄ and R₅ are the same or different, consisting of a hydrogen or alinear or branched alkyl consisting of 1 to 6 carbons,or pharmaceutically acceptable salts thereof.

What are also described herein are any of the compounds listed inATX-001 to ATX-017, ATX-021 to ATX-023, and ATX-026 to ATX-030 listed inTable 1, below, or a pharmaceutically acceptable salt thereof, in alipid composition, comprising a nanoparticle or a bilayer of lipidmolecules. The lipid bilayer preferably further comprises a neutrallipid or a polymer. The lipid composition preferably comprises a liquidmedium. The composition preferably further encapsulates a nucleic acid.The nucleic acid preferably has an activity of suppressing theexpression of the target gene by utilizing RNA interference (RNAi). Thelipid composition preferably further comprises a nucleic acid and aneutral lipid or a polymer. The lipid composition preferablyencapsulates the nucleic acid.

What is also described herein is a compound of Formula II

in whichR₁ and R₂ both consist of a linear alkyl consisting of 1 to 12 carbons,an alkenyl or alkynyl consisting of 2 to 12 carbons,L₁ and L₂ both consist of a linear alkylene or alkenylene consisting of5 to 18 carbons, or forming a heterocycle with N,X is S,L₃ is a bond or a linear alkylene consisting of 1 to 6 carbons, orforming a heterocycle with N,R₃ is a linear or branched alkylene consisting of 1 to 6 carbons, andR₄ and R₅ are the same or different, each a hydrogen or a linear orbranched alkyl consisting of 1 to 6 carbons; or a pharmaceuticallyacceptable salt.

In one embodiment of the compound of formula II, L₁ and L₂ both consistof a linear alkylene consisting of five carbons. In another embodimentof the compound of formula I, R₃ is ethylene or propylene. In anotherembodiment of the compound of formula I, R₄ and R₅ are the same ordifferent, each hydrogen, methyl, or ethyl. In another embodiment of thecompound of formula I, L₃ is a bond. In another embodiment of thecompound of formula I, R₁ and R₂ both consist of a linear alkenylconsisting of ten carbons. In another embodiment of the compound offormula I, the compound consists of a compound selected from formulas 1to 12.

Number Structure 1

2

3

4

5

6

7

8

9

10

11

12

What is also described herein is a compound of formula III or IV

whereinR₁ consists of a branched alkyl with 12 to 20 carbons,R₂ consists of a linear alkyl with 5 to 10 carbons or a branched alkylwith 12 to 20 carbons,L₁ and L₂ each consists of a bond or a linear alkyl having 1 to 3carbons,X consists of S or O,L₃ consists of a bond or a lower alkyl,R₃ consists of a lower alkyl, andR₄ and R₅ are the same or different, each consisting of a lower alkyl;or a pharmaceutically acceptable salt thereof.

In one embodiment of the compound of formula III or IV, L₃ consists of abond. Another embodiment of the compound of formula III or IV, X is S.In another embodiment the compound of formula III or IV, R₃ is ethylene.In another embodiment of the compound of formula III or IV, R₃ isn-propylene or isopropylene. In another embodiment of the compound offormula III or IV, R₄ and R₅ are separately methyl, ethyl, or isopropyl.In another embodiment of the compound of formula III or IV, L₁ and L₂each consists of a bond. In another embodiment of the compound offormula III or IV, L₁ and L₂ consist of a methylene. In anotherembodiment of the compound of formula III or IV, R₁ and R₂ each consistof branched alkyl. In another embodiment of the compound of formula IIIor IV, R₂ consists of an alkyl. In another embodiment of the compound offormula III or IV, R₁ and R₂ each consists of 19 or 20 carbons. Inanother embodiment of the compound of formula III or IV, R₁ or R₂ eachconsists of 13 or 14 carbons. In another embodiment of the compound offormula III or IV, L3 is methylene, R₃ is ethylene, X₂ is S, and R₄ andR₅ are each methyl. In another embodiment of the compound of formula IIIor IV, wherein L₃ is a bond, R₃ is ethylene, X is S, and R₄ and R₅ areeach methyl. In another embodiment of the compound of formula III or IV,L₃ is a bond, R₃ is n-propylene, Xis S, and R₄ and R₅ are each methyl.In another embodiment of the compound of formula III or IV, L₃ is abond, R₃ is isopropylene, X is S, and R₄ and R₅ are each methyl. Inanother embodiment of the compound of formula III or IV, selected fromthe group consisting of a compound of formula ATX-B-1 to ATX-B-12 asfollows.

The compounds of formulas I, II, III, and IV form may be apharmaceutically acceptable salt thereof, in a lipid composition,comprising a nanoparticle or a bilayer of lipid molecules. The lipidbilayer preferably further comprises a neutral lipid or a polymer. Thelipid composition preferably comprises a liquid medium. The compositionpreferably further encapsulates a nucleic acid. The nucleic acidpreferably has an activity of suppressing the expression of the targetgene by utilizing RNA interference (RNAi). The lipid compositionpreferably further comprises a nucleic acid and a neutral lipid or apolymer. The lipid composition preferably encapsulates the nucleic acid.

Reference to compounds of formulas I, II, III, and IV herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic saltsformed with inorganic and/or organic acids, as well as basic saltsformed with inorganic and/or organic bases. In addition, when compoundsof formulas I, II, III, and IV contain both a basic moiety, such as, butnot limited to, a pyridine or imidazole, and an acidic moiety, such as,but not limited to, a carboxylic acid, zwitterions (“inner salts”) maybe formed and are included within the term “salt(s)” as used herein. Thesalts can be pharmaceutically acceptable (i.e., non-toxic,physiologically acceptable) salts, although other salts are also useful.Salts of the compounds of formulas I, II, III, and IV may be formed, forexample, by reacting compounds of formulas I, II, III, and IV with anamount of acid or base, such as an equivalent amount, in a medium suchas one in which the salt precipitates or in an aqueous medium followedby lyophilization.

Exemplary acid addition salts include acetates, adipates, alginates,ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates,borates, butyrates, citrates, camphorates, camphorsulfonates,cyclopentanepropionates, digluconates, dodecylsulfates,ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates,hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides,hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates,methanesulfonates, 2-napthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates,sulfonates (such as those mentioned herein), tartarates, thiocyanates,toluenesulfonates (also known as tosylates) undecanoates, and the like.Additionally, acids which are generally considered suitable for theformation of pharmaceutically useful salts from basic pharmaceuticalcompounds are discussed, for example, by S. Berge et al, J.Pharmaceutical Sciences (1977) 66(1)1-19; P. Gould, International J.Pharmaceutics (1986) 33 201-217; Anderson et al., The Practice ofMedicinal Chemistry (1996), Academic Press, New York; and in The OrangeBook (Food & Drug Administration, Washington, D.C. on their website).These disclosures are incorporated by reference herein.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salts with organic bases (for example,organic amines) such as benzathines, dicyclohexylamines, hydrabamines(formed with N,N-bis(dehydroabietyl)ethylenediamine),N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and saltswith amino acids such as arginine or lysine. Basic nitrogen-containinggroups may be quarternized with agents such as lower alkyl halides(e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, andiodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamylsulfates), long chain halides (e.g., decyl, lauryl, myristyl, andstearyl chlorides, bromides, and iodides), arylalkyl halides (e.g.,benzyl and phenethyl bromides), and others.

All such acid and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the disclosure and all acid andbase salts are considered equivalent to the free forms of thecorresponding compounds of formulas I, II, III, and IV for purposes ofthe disclosure.

Compounds of formulas I, II, III, and IV can exist in unsolvated andsolvated forms, including hydrated forms. In general, the solvatedforms, with pharmaceutically acceptable solvents such as water, ethanol,and the like, are equivalent to the unsolvated forms for the purposes ofthis disclosure.

Compounds of formulas I, II, III, and IV and salts, solvates thereof,may exist in their tautomeric form (for example, as an amide or iminoether). All such tautomeric forms are contemplated herein as part of thepresent disclosure.

Also within the scope of the present disclosure are polymorphs of thecompounds of this disclosure (i.e., polymorphs of the compounds offormulas I, II, III, and IV are within the scope of this disclosure).

All stereoisomers (for example, geometric isomers, optical isomers, andthe like) of the present compounds (including those of the salts,solvates, and prodrugs of the compounds as well as the salts andsolvates of the prodrugs), such as those which may exist due toasymmetric carbons on various substituents, including enantiomeric forms(which may exist even in the absence of asymmetric carbons), rotamericforms, atropisomers, and diastereomeric forms, are contemplated withinthe scope of this disclosure. Individual stereoisomers of the compoundsof this disclosure may, for example, be substantially free of otherisomers, or may be admixed, for example, as racemates or with all other,or other selected, stereoisomers. The chiral centers of the compoundsherein can have the S or R configuration as defined by the IUPAC 1974Recommendations. The use of the terms “salt”, “solvate”, and the like,is intended to equally apply to the salt and solvate of enantiomers,stereoisomers, rotamers, tautomers, racemates, or prodrugs of thedisclosed compounds.

Classes of compounds that can be used as the chemotherapeutic agent(antineoplastic agent) include: alkylating agents, antimetabolites,natural products and their derivatives, hormones and steroids (includingsynthetic analogs), and synthetics. Examples of compounds within theseclasses are given below.

Lipid Particles

The description provides lipid particles comprising one or moretherapeutic mRNA molecules encapsulated within the lipid particles.

In some embodiments, the mRNA is fully encapsulated within the lipidportion of the lipid particle such that the mRNA in the lipid particleis resistant in aqueous solution to nuclease degradation. In otherembodiments, the lipid particles described herein are substantiallynon-toxic to mammals such as humans. The lipid particles typically havea mean diameter of from 30 nm to 150 nm, from 40 nm to 150 nm, from 50nm to 150 nm, from 60 nm to 130 nm, from 70 nm to 110 nm, or from 70 to90 nm. The lipid particles of the invention also typically have alipid:RNA ratio (mass/mass ratio) of from 1:1 to 100:1, from 1:1 to50:1, from 2:1 to 25:1, from 3:1 to 20:1, from 5:1 to 15:1, or from 5:1to 10:1, or from 10:1 to 14:1, or from 9:1 to 20:1. In one embodiment,the lipid particles have a lipid: RNA ratio (mass/mass ratio) of 12:1.In another embodiment, the lipid particles have a lipid: mRNA ratio(mass/mass ratio) of 13:1.

In preferred embodiments, the lipid particles comprise an mRNA, acationic lipid (e.g., one or more cationic lipids or salts thereofdescribed herein), a phospholipid, and a conjugated lipid that inhibitsaggregation of the particles (e.g., one or more PEG-lipid conjugates).The lipid particles can also include cholesterol. The lipid particlesmay comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mRNA thatexpress one or more polypeptides.

In the nucleic acid-lipid particles the mRNA may be fully encapsulatedwithin the lipid portion of the particle, thereby protecting the nucleicacid from nuclease degradation. In preferred embodiments, a lipidparticle comprising an mRNA is fully encapsulated within the lipidportion of the particle, thereby protecting the nucleic acid fromnuclease degradation. In certain instances, the mRNA in the lipidparticle is not substantially degraded after exposure of the particle toa nuclease at 37° C. for at least 20, 30, 45, or 60 minutes. In certainother instances, the mRNA in the lipid particle is not substantiallydegraded after incubation of the particle in serum at 37° C. for atleast 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In otherembodiments, the mRNA is complexed with the lipid portion of theparticle. One of the benefits of the formulations of the presentinvention is that the nucleic acid-lipid particle compositions aresubstantially non-toxic to mammals such as humans.

“Fully encapsulated” means that the nucleic acid (e.g., mRNA) in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free RNA.When fully encapsulated, preferably less than 25% of the nucleic acid inthe particle is degraded in a treatment that would normally degrade 100%of free nucleic acid, more preferably less than 10%, and most preferablyless than 5% of the nucleic acid in the particle is degraded. “Fullyencapsulated” also means that the nucleic acid-lipid particles do notrapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Encapsulation is determined by adding the dye to a liposomalformulation, measuring the resulting fluorescence, and comparing it tothe fluorescence observed upon addition of a small amount of nonionicdetergent. Detergent-mediated disruption of the liposomal bilayerreleases the encapsulated nucleic acid, allowing it to interact with themembrane-impermeable dye. Nucleic acid encapsulation may be calculatedas E=(I₀−I)/I₀, where/and I₀ refers to the fluorescence intensitiesbefore and after the addition of detergent.

In other embodiments, the present invention provides a nucleicacid-lipid particle composition comprising a plurality of nucleicacid-lipid particles.

The lipid particle comprises mRNA that is fully encapsulated within thelipid portion of the particles, such that from 30% to 100%, from 40% to100%, from 50% to 100%, from 60% to 100%, from 70% to 100%, from 80% to100%, from 90% to 100%, from 30% to 95%, from 40% to 95%, from 50% to95%, from 60% to 95%, from 70% to 95%, from 80% to 95%, from 85% to 95%,from 90% to 95%, from 30% to 90%, from 40% to 90%, from 50% to 90%, from60% to 90%, from 70% to 90%, from 80% to 90%, or at least 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% (or any fraction thereof or range therein) ofthe particles have the mRNA encapsulated therein.

Depending on the intended use of the lipid particles, the proportions ofthe components can be varied and the delivery efficiency of a particularformulation can be measured using assays know in the art.

Cationic Lipids

The description includes synthesis of certain cationic lipid compounds.The compounds are particularly suitable for delivering polynucleotidesto cells and tissues as demonstrated in subsequent sections. Thelipomacrocycle compound described herein may be used for other purposesas well as, for example, recipients and additives.

The synthetic methods for the cationic lipid compounds can besynthesized with the skills in the art. The skilled of the art willrecognize other methods to produce these compounds, and to produce alsothe other compounds of the description.

The cationic lipid compounds may be combined with an agent to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The lipomacrocycle compounds may be combinedwith other cationic lipid compounds, polymers (synthetic or natural),surfactants, cholesterol, carbohydrates, proteins, or lipids, to formthe particles. These particles may then optionally be combined with apharmaceutical excipient to form a pharmaceutical composition.

The present description provides novel cationic lipid compounds and drugdelivery systems based on the use of such cationic lipid compounds. Thesystem may be used in the pharmaceutical/drug delivery arts to deliverpolynucleotides, proteins, small molecules, peptides, antigen, or drugs,to a patient, tissue, organ, or cell. These novel compounds may also beused as materials for coating, additives, excipients, materials, orbioengineering.

The cationic lipid compounds of the present description provide forseveral different uses in the drug delivery art. The amine-containingportion of the cationic lipid compounds may be used to complexpolynucleotides, thereby enhancing the delivery of polynucleotide andpreventing their degradation. The cationic lipid compounds may also beused in the formation of picoparticles, nanoparticles, microparticles,liposomes, and micelles containing the agent to be delivered.Preferably, the cationic lipid compounds are biocompatible andbiodegradable, and the formed particles are also biodegradable andbiocompatible and may be used to provide controlled, sustained releaseof the agent to be delivered. These and their corresponding particlesmay also be responsive to pH changes given that these are protonated atlower pH. They may also act as proton sponges in the delivery of anagent to a cell to cause endosome lysis.

In certain embodiments, the cationic lipid compounds are relativelynon-cytotoxic. The cationic lipid compounds may be biocompatible andbiodegradable. The cationic lipid may have a pK_(a) in the range ofapproximately 5.5 to approximately 7.5, more preferably betweenapproximately 6.0 and approximately 7.0. It may be designed to have adesired pK_(a) between approximately 3.0 and approximately 9.0, orbetween approximately 5.0 and approximately 8.0. The cationic lipidcompounds described herein are particularly attractive for drug deliveryfor several reasons: they contain amino groups for interacting with DNA,RNA, other polynucleotides, and other negatively charged agents, forbuffering the pH, for causing endo-osmolysis, for protecting the agentto be delivered, they can be synthesized from commercially availablestarting materials; and/or they are pH responsive and can be engineeredwith a desired pK_(a).

A composition containing a cationic lipid compound may be 30-70%cationic lipid compound, 0-60% cholesterol, 0-30% phospholipid and 1-10%polyethylene glycol (PEG). Preferably, the composition is 30-40%cationic lipid compound, 40-50% cholesterol, and 10-20% PEG. In otherpreferred embodiments, the composition is 50-75% cationic lipidcompound, 20-40% cholesterol, and 5-10% phospholipid, and 1-10% PEG. Thecomposition may contain 60-70% cationic lipid compound, 25-35%cholesterol, and 5-10% PEG. The composition may contain up to 90%cationic lipid compound and 2-15% helper lipid.

The formulation may be a lipid particle formulation, for examplecontaining 8-30% compound, 5-30% helper lipid, and 0-20% cholesterol;4-25% cationic lipid, 4-25% helper lipid, 2-25% cholesterol, 10-35%cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipid,2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20%cholesterol-amine; or up to 90% cationic lipid and 2-10% helper lipids,or even 100% cationic lipid.

Non-Cationic Lipids

The non-cationic lipids that are used in lipid particles can be any of avariety of neutral uncharged, zwitterionic, or anionic lipids capable ofproducing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5α-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5α-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether.

In some embodiments, the non-cationic lipid present in lipid particlescomprises or consists of a mixture of one or more phospholipids andcholesterol or a derivative thereof. In other embodiments, thenon-cationic lipid present in the lipid particles comprises or consistsof one or more phospholipids, e.g., a cholesterol-free lipid particleformulation. In yet other embodiments, the non-cationic lipid present inthe lipid particles comprises or consists of cholesterol or a derivativethereof, e.g., a phospholipid-free lipid particle formulation.

Other examples of non-cationic lipids include nonphosphorous containinglipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetylpalmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate,amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-arylsulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, and sphingomyelin.

In some embodiments, the non-cationic lipid comprises from 10 mol % to60 mol %, from 20 mol % to 55 mol %, from 20 mol % to 45 mol %, 20 mol %to 40 mol %, from 25 mol % to 50 mol %, from 25 mol % to 45 mol %, from30 mol % to 50 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol%, from 35 mol % to 45 mol %, from 37 mol % to 42 mol %, or 35 mol %, 36mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43mol %, 44 mol %, or 45 mol % (or any fraction thereof or range therein)of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol %of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from 2 mol % to 20 mol %, from 2 mol % to 15 mol %, from 2 mol% to 12 mol %, from 4 mol % to 15 mol %, or from 4 mol % to 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from 5 mol % to 10 mol %, from 5 mol % to 9 mol%, from 5 mol % to 8 mol %, from 6 mol % to 9 mol %, from 6 mol % to 8mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle.

In other embodiments, the cholesterol component in the mixture maycomprise from 25 mol % to 45 mol %, from 25 mol % to 40 mol %, from 30mol % to 45 mol %, from 30 mol % to 40 mol %, from 27 mol % to 37 mol %,from 25 mol % to 30 mol %, or from 35 mol % to 40 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Incertain preferred embodiments, the cholesterol component in the mixturecomprises from 25 mol % to 35 mol %, from 27 mol % to 35 mol %, from 29mol % to 35 mol %, from 30 mol % to 35 mol %, from 30 mol % to 34 mol %,from 31 mol % to 33 mol %, or 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, or 35 mol % (or any fraction thereof or range therein) of thetotal lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to 25 mol %, 30 mol %,35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of thetotal lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from 25 mol %to 45 mol %, from 25 mol % to 40 mol %, from 30 mol % to 45 mol %, from30 mol % to 40 mol %, from 31 mol % to 39 mol %, from 32 mol % to 38 mol%, from 33 mol % to 37 mol %, from 35 mol % to 45 mol %, from 30 mol %to 35 mol %, from 35 mol % to 40 mol %, or 30 mol %, 31 mol %, 32 mol %,33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or40 mol % (or any fraction thereof or range therein) of the total lipidpresent in the particle.

In other embodiments, the non-cationic lipid comprises from 5 mol % to90 mol %, from 10 mol % to 85 mol %, from 20 mol % to 80 mol %, 10 mol %(e.g., phospholipid only), or 60 mol (e.g., phospholipid and cholesterolor derivative thereof) (or any fraction thereof or range therein) of thetotal lipid present in the particle.

The percentage of non-cationic lipid present in the lipid particles is atarget amount, and that the actual amount of non-cationic lipid presentin the formulation may vary, for example, by ±5 mol %.

Lipid Conjugates

In addition to cationic, the lipid particles described herein mayfurther comprise a lipid conjugate. The conjugated lipid is useful inthat it prevents the aggregation of particles. Suitable conjugatedlipids include, but are not limited to, PEG-lipid conjugates,cationic-polymer-lipid conjugates, and mixtures thereof.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA), PEG coupled to diacylglycerol (PEG-DAG),PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE),PEG conjugated to ceramides, PEG conjugated to cholesterol or aderivative thereof, and mixtures thereof.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; and include the following: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH₂).

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from 550 daltons to 10,000 daltons.In certain instances, the PEG moiety has an average molecular weight offrom 750 daltons to 5,000 daltons (e.g., from 1,000 daltons to 5,000daltons, from 1,500 daltons to 3,000 daltons, from 750 daltons to 3,000daltons, from 750 daltons to 2,000 daltons). In preferred embodiments,the PEG moiety has an average molecular weight of 2,000 daltons or 750daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester-containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is anon-ester-containing linker moiety. Suitable non-ester-containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (-0-), succinyl (-(0)CCH₂CH₂C(0)-),succinamidyl (—NHC(0)CH₂CH₂C(0)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester-containing linker moiety is used tocouple the PEG to the lipid. Suitable ester-containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skill in the art.Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or di-unsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoyl-phosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl).

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R and R, both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation.

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of 750or 2,000 daltons. In particular embodiments, the terminal hydroxyl groupof the PEG is substituted with a methyl group.

In addition to the foregoing, other hydrophilic polymers can be used inplace of PEG. Examples of suitable polymers that can be used in place ofPEG include, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom 0.1 mol % to 2 mol %, from 0.5 mol % to 2 mol %, from 1 mol % to 2mol %, from 0.6 mol % to 1.9 mol %, from 0.7 mol % to 1.8 mol %, from0.8 mol % to 1.7 mol %, from 0.9 mol % to 1.6 mol %, from 0.9 mol % to1.8 mol %, from 1 mol % to 1.8 mol %, from 1 mol % to 1.7 mol %, from1.2 mol % to 1.8 mol %, from 1.2 mol % to 1.7 mol %, from 1.3 mol % to1.6 mol %, or from 1.4 mol % to 1.5 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle. In otherembodiments, the lipid conjugate (e.g., PEG-lipid) comprises from 0 mol% to 20 mol %, from 0.5 mol % to 20 mol %, from 2 mol % to 20 mol %,from 1.5 mol % to 18 mol %, from 2 mol % to 15 mol %, from 4 mol % to 15mol %, from 2 mol % to 12 mol %, from 5 mol % to 12 mol %, or 2 mol %(or any fraction thereof or range therein) of the total lipid present inthe particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom 4 mol % to 10 mol %, from 5 mol % to 10 mol %, from 5 mol % to 9mol %, from 5 mol % to 8 mol %, from 6 mol % to 9 mol %, from 6 mol % to8 mol %, or 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle.

The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipidparticles of the invention is a target amount, and the actual amount oflipid conjugate present in the formulation may vary, for example, by ±2mol %. One of ordinary skill in the art will appreciate that theconcentration of the lipid conjugate can be varied depending on thelipid conjugate employed and the rate at which the lipid particle is tobecome fusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. In addition, other variables including, e.g., pH,temperature, or ionic strength, can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle size.

Compositions and Formulations for Administration

The nucleic acid-lipid compositions of this disclosure may beadministered by various routes, for example, to effect systemic deliveryvia intravenous, parenteral, intraperitoneal, or topical routes. In someembodiments, a siRNA may be delivered intracellularly, for example, incells of a target tissue such as lung or liver, or in inflamed tissues.In some embodiments, this disclosure provides a method for delivery ofsiRNA in vivo. A nucleic acid-lipid composition may be administeredintravenously, subcutaneously, or intraperitoneally to a subject. Insome embodiments, the disclosure provides methods for in vivo deliveryof interfering RNA to the lung of a mammalian subject.

In some embodiments, this disclosure provides a method of treating adisease or disorder in a mammalian subject. A therapeutically effectiveamount of a composition of this disclosure containing a nucleic, acationic lipid, an amphiphile, a phospholipid, cholesterol, and aPEG-linked cholesterol may be administered to a subject having a diseaseor disorder associated with expression or overexpression of a gene thatcan be reduced, decreased, downregulated, or silenced by thecomposition.

The compositions and methods of the disclosure may be administered tosubjects by a variety of mucosal administration modes, including byoral, rectal, vaginal, intranasal, intrapulmonary, or transdermal ordermal delivery, or by topical delivery to the eyes, ears, skin, orother mucosal surfaces. In some aspects of this disclosure, the mucosaltissue layer includes an epithelial cell layer. The epithelial cell canbe pulmonary, tracheal, bronchial, alveolar, nasal, buccal, epidermal,or gastrointestinal. Compositions of this disclosure can be administeredusing conventional actuators such as mechanical spray devices, as wellas pressurized, electrically activated, or other types of actuators.

Compositions of this disclosure may be administered in an aqueoussolution as a nasal or pulmonary spray and may be dispensed in sprayform by a variety of methods known to those skilled in the art.Pulmonary delivery of a composition of this disclosure is achieved byadministering the composition in the form of drops, particles, or spray,which can be, for example, aerosolized, atomized, or nebulized.Particles of the composition, spray, or aerosol can be in either aliquid or solid form. Preferred systems for dispensing liquids as anasal spray are disclosed in U.S. Pat. No. 4,511,069. Such formulationsmay be conveniently prepared by dissolving compositions according to thepresent disclosure in water to produce an aqueous solution, andrendering said solution sterile. The formulations may be presented inmulti-dose containers, for example in the sealed dispensing systemdisclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spraydelivery systems have been described in Transdermal Systemic Medication,Y. W. Chien ed., Elsevier Publishers, New York, 1985; and in U.S. Pat.No. 4,778,810. Additional aerosol delivery forms may include, e.g.,compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, whichdeliver the biologically active agent dissolved or suspended in apharmaceutical solvent, e.g., water, ethanol, or mixtures thereof.

Nasal and pulmonary spray solutions of the present disclosure typicallycomprise the drug or drug to be delivered, optionally formulated with asurface active agent, such as a nonionic surfactant (e.g.,polysorbate-80), and one or more buffers. In some embodiments of thepresent disclosure, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution may be from pH 6.8 to7.2. The pharmaceutical solvents employed can also be a slightly acidicaqueous buffer of pH 4-6. Other components may be added to enhance ormaintain chemical stability, including preservatives, surfactants,dispersants, or gases.

In some embodiments, this disclosure is a pharmaceutical product whichincludes a solution containing a composition of this disclosure and anactuator for a pulmonary, mucosal, or intranasal spray or aerosol.

A dosage form of the composition of this disclosure can be liquid, inthe form of droplets or an emulsion, or in the form of an aerosol.

A dosage form of the composition of this disclosure can be solid, whichcan be reconstituted in a liquid prior to administration. The solid canbe administered as a powder. The solid can be in the form of a capsule,tablet, or gel.

To formulate compositions for pulmonary delivery within the presentdisclosure, the biologically active agent can be combined with variouspharmaceutically acceptable additives, as well as a base or carrier fordispersion of the active agent(s). Examples of additives include pHcontrol agents such as arginine, sodium hydroxide, glycine, hydrochloricacid, citric acid, and mixtures thereof. Other additives include localanesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodiumchloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),solubility enhancing agents (e.g., cyclodextrins and derivativesthereof), stabilizers (e.g., serum albumin), and reducing agents (e.g.,glutathione). When the composition for mucosal delivery is a liquid, thetonicity of the formulation, as measured with reference to the tonicityof 0.9% (w/v) physiological saline solution taken as unity, is typicallyadjusted to a value at which no substantial, irreversible tissue damagewill be induced in the mucosa at the site of administration. Generally,the tonicity of the solution is adjusted to a value of ⅓ to 3, moretypically ½ to 2, and most often ¾ to 1.7.

The biologically active agent may be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base may be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (e.g., maleic anhydride) with other monomers (e.g.,methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymerssuch as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives such as hydroxymethylcellulose,hydroxypropylcellulose, etc., and natural polymers such as chitosan,collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metalsalts thereof. Often, a biodegradable polymer is selected as a base orcarrier, for example, polylactic acid, poly(lactic acid-glycolic acid)copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolicacid) copolymer, and mixtures thereof. Alternatively or additionally,synthetic fatty acid esters such as polyglycerin fatty acid esters,sucrose fatty acid esters, etc., can be employed as carriers.Hydrophilic polymers and other carriers can be used alone or incombination, and enhanced structural integrity can be imparted to thecarrier by partial crystallization, ionic bonding, crosslinking, and thelike. The carrier can be provided in a variety of forms, including fluidor viscous solutions, gels, pastes, powders, microspheres, and films fordirect application to the nasal mucosa. The use of a selected carrier inthis context may result in promotion of absorption of the biologicallyactive agent.

Formulations for mucosal, nasal, or pulmonary delivery may contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10,000 and preferably not more than3,000. Examples of hydrophilic low molecular weight compounds includepolyol compounds, such as oligo-, di- and monosaccarides includingsucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose,D-xylose, D-mannose, D-galactose, lactulose, cellobiose, gentibiose,glycerin, polyethylene glycol, and mixtures thereof. Further examples ofhydrophilic low molecular weight compounds include N-methylpyrrolidone,alcohols (e.g., oligovinyl alcohol, ethanol, ethylene glycol, propyleneglycol, etc.), and mixtures thereof.

The compositions of this disclosure may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, and wetting agents, for example,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, andmixtures thereof. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

In certain embodiments of the disclosure, the biologically active agentmay be administered in a time release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system, or bioadhesive gel. Prolongeddelivery of the active agent, in various compositions of the disclosurecan be brought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.

While this disclosure has been described in relation to certainembodiments, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that thisdisclosure includes additional embodiments, and that some of the detailsdescribed herein may be varied considerably without departing from thisdisclosure. This disclosure includes such additional embodiments,modifications, and equivalents. In particular, this disclosure includesany combination of the features, terms, or elements of the variousillustrative components and examples.

EXAMPLES Example 1

Exemplary compounds of formula I are provided in Table 1.

TABLE 1 KD @ Lipid 0.3 ID Novel Lipid MW pKa mg/kg ATX- 001

695.1 8.9 ~0 ATX- 002

681 8.7 98 ATX- 003

695.1 9.3 ~0 ATX- 004

709.13 9.4 ~0 ATX- 005

709.13 9.0 ~0 ATX- 006

723.15 9.8 ~0 ATX- 007

723.15 9.5 n/a ATX- 008

737.18 10.3 n/a ATX- 009

695.1 8.8 ~0 ATX- 010

709.13 9.6 30 ATX- 011

709.13 9.4 n/a ATX- 012

723.15 10.2 ~0 ATX- 013

681.01 n/a ATX- 014

695.1 n/a ATX- 015

695.1 n/a ATX- 016

709.13 15 ATX- 017

695.1 n/a ATX- 021

679.04 n/a ATX- 022

665.01 n/a ATX- 023

695.1 n/a ATX- 026

681.07 n/a ATX- 027

695.1 n/a ATX- 028

681.07 n/a ATX- 029

681.1 n/a ATX- 030

695.1 n/aTable 1 shows the name and structure of each compound, its molecularweight, its pKa, and its knockdown bioactivity (KD) in an assaydescribed below in Example 19.

Exemplary compounds of formulas II and III are provided in Tables 2, and3.

TABLE 2 Number Structure 1

2

3

4

5

6

7

8

9

10

11

12

TABLE 3 Name Structure ATX-B-1

ATX-B-2

ATX-B-3

ATX-B-4

ATX-B-5

ATX-B-6

ATX-B-7

ATX-B-8

ATX-B-9

ATX-B-10

ATX-B-12

Example 2 Synthesis of methyl 8-bromooctanoate

Under N₂ atmosphere, 8-bromooctanoic acid was dissolved in dry methanol.Concentrated H₂SO₄ was added drop-wise and the reaction mixture wasstirred under reflux for three hours.

The reaction was monitored by thin layer chromatography until completed.Solvent was completely removed under vacuum. The reaction mixture wasdiluted with ethyl acetate and washed with water. The water layer wasre-extracted with ethyl acetate. The total organic layer was washed witha saturated NaHCO₃ solution. The organic layer was washed again withwater and finally washed with brine. The product was dried overanhydrous Na₂SO₄ and concentrated.

Example 3 Synthesis of dimethyl 8,8′-(benzanediyl)dioctanoate

Dry K₂CO₃ was taken and added to dry dimethylformamide under N₂. Benzylamine in dimethylformamide was slowly added. Methyl 8-bromooctanoatedissolved in dimethylformamide was then added at room temperature. Thereaction mixture was heated to 80° C. and the reaction was maintainedfor 36 hours with stirring.

The reaction was monitored by thin layer chromatography until completed.The reaction product was cooled to room temperature and water was added.The compound was extracted with ethyl acetate. The water layer wasre-extracted with ethyl acetate. The total organic layer was washed withwater and finally with brine solution. The product was dried overanhydrous Na₂SO₄ and concentrated.

The reaction product was purified by silica gel column chromatography in3% methanol in chloroform 44 grams (g) of pure product was recovered.

Using thin layer chromatograph (TLC) system of 10% methanol inchloroform, the product migrated with a Rf of 0.8, visualizing bycharring in ninhydrine. The overall yield was 82%. The compound was alight brown liquid. The structure was confirmed by ¹H-NMR.

Example 4 Synthesis of dimethyl 8,8′-azanediyldioctanoate

Dimethyl 8,8′-(benzanediyl)dioctanoate was transferred to hydrogenationglass vessel, and ethanol was added followed by 10% Pd/C. The reactionmixture was shaken in a Parr-shaker apparatus under 50 pounds per squareinch (psi) H₂ atmosphere pressure for two hours at room temperature.

The reaction product was filtered through celite and washed with hotethyl acetate. The filtrate was concentrated under vacuum.

Example 5 Synthesis of dimethyl 8,8′-((tertbutoxycarbonyl)azanedil)dioctanoate

Dimethyl 8,8′-azanediyldioctanoate was transferred to dichloromethane(DCM) and Et₃N to the reaction mass and cooled to 0° C. Boc anhydridediluted in DCM was added drop to the above reaction. After the additionwas completed, the reaction mixture was stirred at room temperature forthree hours.

The reaction was quenched with water and the DCM layer was separated.The water phase was re-extracted with DCM and the combined DCM layerswere washed with brine solution and dried with Na₂SO₄. Afterconcentration, 40 g of crude compound was collected.

Crude reaction product was purified by column chromatography using 0-12%ethyl acetate in hexane. The yield recovered was 48%. A single productmigrated by thin layer chromatography in 20% ethyl acetate in hexanewith an Rf of 0.5, charring with ninhydrine.

Example 6 Synthesis of 8,8′-((tertbutoxycarbonyl)azanediyl) dioctanoicacid

Dimethyl 8,8′-((tertbutoxycarbonyl)azanedil) dioctanoate was transferredto tetrahydrofuran (THF). A 6N sodium hydroxide solution was added atroom temperature. The reaction was maintained with stirring overnight atroom temperature.

Reaction mass was evaporated under vacuum at 25° C. to remove THF. Thereaction product was acidified with 5N HCl. Ethyl acetate was added tothe aqueous layer. The separated organic layer was washed with water andthe water layer was re-extracted with ethyl acetate. The combinedorganic layers were washed with brine solution and dried over anhydrousNa₂SO₄. Concentration of the solution gave 18 g of crude mass.

Example 7 Synthesis of di((Z)-non-2-en-1-yl)8,8′((tertbutoxycarbonyl)azanediyl)

8,8′-((tertbutoxycarbonyl)azanediyl) dioctanoic acid was dissolved indry DCM.1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) was added to this solution.Di-isopropyl ethyl amine was added slowly to the reaction mixture atroom temperature. The internal temp rose to 40° C. and a pale yellowcolor solution was formed. 4-Dimethylaminopyridine (DMAP) was added tothe reaction mixture followed by cis-2-nonene-1-ol solution in dry DCM.The reaction changed to brown color. The reaction was stirred for fivehours at room temperature.

The reaction was checked by thin layer chromatography under completion.Water was added to the reaction product, which was extracted with DCM.The DCM layer was washed with water followed by brine solution. Theorganic layer was dried over anhydrous Na₂SO₄ and concentrated to obtain35 g of crude compound.

Example 8 Synthesis of ATX-001

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.023 mol, 15 g) was dissolved in dry dichloromethane (DCM) (200 ml).Trifluoroacetic acid (TFA) was added at 0° C. to initiate a reaction.The reaction temperature was slowly allowed to warm to room temperaturefor 30 minutes with stirring. Thin layer chromatography showed that thereaction was completed. The reaction product was concentrated undervacuum at 40° C. and the crude residue was diluted with DCM, and washedwith a 10% NaHCO₃ solution. The aqueous layer was re-extracted with DCM,and the combined organic layers were washed with brine solution, driedover Na₂SO₄ and concentrated. The collected crude product (12 grams) wasdissolved in dry DCM (85 ml) under nitrogen gas. Triphosgene were addedand the reaction mixture was cooled to 0° C., and Et₃N was added dropwise. The reaction mixture was stirred overnight at room temperature.Thin layer chromatography showed that the reaction was completed. DCMsolvent was removed from the reaction mass by distillation under N₂. Thereaction product was cooled to 0° C., diluted with DCM (50 ml), and2-((2-(dimethylamino)ethyl)thio) acetic acid (0.039 mol, 6.4 g) andcarbodiimide (EDC HCl) (0.054 mol, 10.4 g). The reaction mixture wasthen stirred overnight at room temperature. Thin layer chromatographyshowed that the reaction was completed. The reaction product was dilutedwith 0.3M HCl solution (75 ml), and the organic layer was separated. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with 10% K₂CO₃ aqueous solution (75 ml) and dried overanhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10gram. The crude compound was purified by silica gel column (100-200mesh) using 3% MeOH/DCM. The yield was 10.5 g (68%).

Example 9 Synthesis of ATX-002

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(13.85 mmol, 9 grams) was dissolved in dry DCM (150 ml). TFA was addedat 0° C. to initiate a reaction. The reaction temperature was slowlyallowed to warm to room temperature for 30 minutes with stirring. Thinlayer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentrated. Thecollected crude product was dissolved in dry DCM (85 ml) under nitrogengas. Triphosgene were added and the reaction mixture was cooled to 0°C., and Et₃N was added drop wise. The reaction mixture was stirredovernight at room temperature. Thin layer chromatography showed that thereaction was completed. DCM solvent was removed from the reaction massby distillation under N₂. The reaction product was cooled to 0° C.,diluted with DCM (50 ml), and 2-(dimethylamino)ethanethiol HCl (0.063mol, 8.3 g) was added, followed by Et₃N (dry). The reaction mixture wasthen stirred overnight at room temperature. Thin layer chromatographyshowed that the reaction was completed. The reaction product was dilutedwith 0.3M HCl solution (75 ml), and the organic layer was separated. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with 10% K₂CO₃ aqueous solution (75 ml) and dried overanhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of 10gram. The crude compound was purified by silica gel column (100-200mesh) using 3% MeOH/DCM. The yield was 3.1 gram.

Example 10 Synthesis of ATX-003

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.00337 mol, 2.2 g) was dissolved in dry DCM (20 ml). TFA was added at0° C. to initiate a reaction. The reaction temperature was slowlyallowed to warm to room temperature for 30 minutes with stirring. Thinlayer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentratedunder reduced pressure. The collected crude product was dissolved in dryDCM (10 ml) under nitrogen gas. Triphosgene (0.0182 mol, 5.4 g) wasadded and the reaction mixture was cooled to 0° C., and Et₃N was addeddrop wise. The reaction mixture was stirred overnight at roomtemperature. Thin layer chromatography showed that the reaction wascompleted. DCM solvent was removed from the reaction mass bydistillation under N₂. The reaction product was cooled to 0° C., dilutedwith DCM (15 ml), and 2-(dimethylamino)propanethiol HCl (0.0182 mol,2.82 g) was added, followed by Et₃N (dry). The reaction mixture was thenstirred overnight at room temperature. Thin layer chromatography showedthat the reaction was completed. The reaction product was diluted with0.3 M HCl aqueous solution (20 ml), and the organic layer was separated.The aqueous layer was re-extracted with DCM, and the combined organiclayers were washed with 10% K₂CO₃ aqueous solution (50 ml) and driedover anhydrous Na₂SO₄. Concentration of the solvent gave a crude mass of5 gram. The crude compound was purified by silica gel column (100-200mesh) using 3% MeOH/DCM. The yield was 0.9 gram.

Example 11 Synthesis of ATX-004

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.023 mol, 15 g) was dissolved in DCM (200 ml). TFA was added at 0° C.to initiate a reaction. The reaction temperature was slowly allowed towarm to room temperature for 30 minutes with stirring. Thin layerchromatography showed that the reaction was completed. The reactionproduct was concentrated under vacuum at 40° C. and the crude residuewas diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueouslayer was re-extracted with DCM, and the combined organic layers werewashed with brine solution, dried over Na₂SO₄ and concentrated. Thecollected crude product, di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate(5.853 mmol, 3.2 g) was dissolved in dry dimethyl formamide (DMF) undernitrogen, and 2-((3-(dimethylamino)propyl)thio)acetic acid (10.48 mmol,1.85 g) and EDC HCl (14.56 mmol, 2.78 g) was added. The reaction mixturewas stirred overnight at room temperature. The reaction was quenchedwith water (30 ml) and diluted with DCM (30 ml), and the organic layerwas separated. The aqueous layer was re-extracted with DCM, and thecombined organic layers were washed with 10% K₂CO₃ aqueous solution anddried over anhydrous Na₂SO₄. The crude compound was purified by silicagel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1 gram(24.2%).

Example 12 Synthesis of ATX-005

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(0.023 mol, 15 g) was dissolved in dry DCM (200 ml). TFA was added at 0°C. to initiate a reaction. The reaction temperature was slowly allowedto warm to room temperature for 30 minutes with stirring. Thin layerchromatography showed that the reaction was completed. The reactionproduct was concentrated under vacuum at 40° C. and the crude residuewas diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueouslayer was re-extracted with DCM, and the combined organic layers werewashed with brine solution, dried over Na₂SO₄ and concentrated. Crudereaction product, di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate (5.853mmol, 3.2 g) was dissolved in dimethylformamide (DMF) under nitrogengas. 2-((3-(dimethylamino)propyl)thio)acetic acid (10.48 mmol, 1.85 g)and EDC HCl (14.56 mmol, 2.78 g) were added and the reaction mixture wasstirred overnight at room temperature. Thin layer chromatography showedthat the reaction was completed. The reaction product was quenched withwater (30 ml) and diluted with DCM (30 ml). The aqueous layer wasre-extracted with DCM, and the combined organic layers were washed with10% K₂CO₃ aqueous solution (75 ml) and dried over anhydrous Na₂SO₄.Concentration of the solvent gave a crude mass of 5 grams. Crudecompound was purified by silica gel column (100-200 mesh) using 3%MeOH/DCM. The yield was 1 gram (24.2%).

Example 13 Synthesis of ATX-006

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoatewas dissolved in dry DCM (150 ml). TFA was added at 0° C. to initiate areaction. The reaction temperature was slowly allowed to warm to roomtemperature for 30 minutes with stirring. Thin layer chromatographyshowed that the reaction was completed. The reaction product wasconcentrated under vacuum at 40° C. and the crude residue was dilutedwith DCM, and washed with a 10% NaHCO₃ solution. The aqueous layer wasre-extracted with DCM, and the combined organic layers were washed withbrine solution, dried over Na₂SO₄ and concentrated. The collected crudeproduct was dissolved in dry DCM (85 ml) under nitrogen gas. Triphosgenewere added and the reaction mixture was cooled to 0° C., and Et₃N wasadded drop wise. The reaction mixture was stirred overnight at roomtemperature. Thin layer chromatography showed that the reaction wascompleted. The crude reaction product was dissolved in dry DMF undernitrogen atmosphere, and 2-((2-(diethylamino)ethyl)thio)acetic acid(3.93 mmol, 751 mg) and EDC HCl (5.45 mmol, 1.0 g) were added. Thereaction mixture was stirred overnight at room temperature. The reactionwas quenched with water (3 ml) and excess DMF was removed under vacuumat 25° C. The reaction product was diluted with water and aqueous layerwas extracted thrice with DCM (20 ml). The combined organic layers werewashed with brine solution and dried over anhydrous Na₂SO₄.Concentration of the solvent gave a crude mass of 2 g. Afterpurification by silica gel column (100-200 mesh) using 3% MeOH/DCM., theyield was 1.2 g (76%).

Example 14 Synthesis of ATX-009

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(13.85 mmol, 9 grams) was dissolved in dry DCM (20 ml). TFA was added at0° C. to initiate a reaction. The reaction temperature was slowlyallowed to warm to room temperature for 30 minutes with stirring. Thinlayer chromatography showed that the reaction was completed. Thereaction product was concentrated under vacuum at 40° C. and the cruderesidue was diluted with DCM, and washed with a 10% NaHCO₃ solution. Theaqueous layer was re-extracted with DCM, and the combined organic layerswere washed with brine solution, dried over Na₂SO₄ and concentrated.Di((Z)-non-2-en-1-yl)8,8′-azanediyldioctanoate (0.909 mmol, 500 mg) wasdissolved in dry DCM (20 ml) under nitrogen atmosphere. Triphosgene wereadded and the reaction mixture was cooled to 0° C., and Et₃N was addeddrop wise. The reaction mixture was stirred overnight at roomtemperature. Thin layer chromatography showed that the reaction wascompleted. DCM solvent was removed from the reaction mass bydistillation under nitrogen atmosphere.2-(ethyl(methyl)amino)ethane-1-thiol hydrochloride (4.575 mmol, 715 mg)was dissolved in DMF (7 ml) and tetrahydrofuran (THF) (5 ml), and wasadded drop wise to the sodium hydride suspension in THF at 0° C. Thereaction mixture was then stirred overnight at room temperature. Thinlayer chromatography showed that the reaction was completed. Thereaction product was diluted with ethyl acetate and cold water. Thereaction was neutralized with 5% HCl (9 ml), and the organic layer wasseparated. The aqueous layer was re-extracted with ethyl acetate (EtOAc)(20 ml), washed in cold water and brine, and the combined organic layerswere washed and dried over anhydrous Na₂SO₄. Concentration of thesolvent gave 1 gram or crude product. The compound was purified bysilica gel column (100-200 mesh) using 3% MeOH/DCM to yield 100 mg.

Example 15 Synthesis of ATX-010

Di((Z)-non-2-en-1-yl) 8,8′((tertbutoxycarbonyl)azanediyl) dioctanoate(3.079 mmol, 2 g) was dissolved in dry DCM (20 ml). TFA was added at 0°C. to initiate a reaction. The reaction temperature was slowly allowedto warm to room temperature for 30 minutes with stirring. Thin layerchromatography showed that the reaction was completed. The reactionproduct was concentrated under vacuum at 40° C. and the crude residuewas diluted with DCM, and washed with a 10% NaHCO₃ solution. The aqueouslayer was re-extracted with DCM, and the combined organic layers werewashed with brine solution, dried over Na₂SO₄ and concentrated. Thecollected crude product was dissolved in dry DCM (20 ml) under nitrogengas. Triphosgene (14.55 mmol, 4.32 g) was added and the reaction mixturewas cooled to 0° C., and Et₃N was added drop wise. The reaction mixturewas stirred overnight at room temperature. Thin layer chromatographyshowed that the reaction was completed. DCM solvent was removed from thereaction mass by distillation under N₂. The reaction product was cooledto 0° C., diluted with DCM (20 ml), and 2-(dimethylamino)ethanethiol HCl(0.063 mol, 8.3 g) was added, followed by Et₃N (dry). The reactionmixture was then stirred overnight at room temperature. Thin layerchromatography showed that the reaction was completed. The reactionproduct was diluted with 0.3 M HCl solution (20 ml), and the organiclayer was separated. The aqueous layer was re-extracted with DCM, andthe combined organic layers were washed with 10% K₂CO₃ aqueous solution20 ml) and dried over anhydrous Na₂SO₄. Concentration of the solventgave a crude mass of 10 gram. The crude compound was purified by silicagel column (100-200 mesh) using 3% MeOH/DCM. The yield was 1.4 g (75%).

Example 16 Synthesis of ATX-011 to ATX-017, ATX-021 to ATX-023, andATX-026 to ATX-030 from Table 1, and the Compounds of Tables 2 and 3

The synthesis of ATX-011 to ATX-017, ATX-021 to ATX-023, and ATX-026 toATX-030 and the compounds of Tables 2 and 3 follows the synthesis ofExamples 1 to 15, by substituting appropriate starting ingredients forsynthetic reactions described therein.

Example 17 In Vivo Mouse Factor VII Silencing

Using a liver-directed in vivo screen of the liposome libraries, aseries of compounds were tested that facilitate high levels of siRNAmediated gene silencing in hepatocytes, the cells comprising the liverparenchyma. Factor VII, a blood clotting factor, is a suitable targetgene for assaying functional siRNA delivery to liver. Because thisfactor is produced specifically in hepatocytes, gene silencing indicatessuccessful delivery to parenchyma, as opposed to delivery to the cellsof the reticulo-endothelial system (e.g., Kupffer cells). Furthermore,Factor VII is a secreted protein that can be readily measured in serum,obviating the need to euthanize animals. Silencing at the mRNA level canbe readily determined by measuring levels of protein. This is becausethe protein's short half-life (2-5 hour). C57BL/6 mice (Charles RiverLabs) received either saline or siRNA in liposome formulations via tailvein injection at a volume of 0.006 ml/g. At 48 h after administration,animals were anesthetized by isofluorane inhalation and blood wascollected into serum separator tubes by retroorbital bleed. Serum levelsof Factor VII protein were determined in samples using a chromogenicassay (Biophen FVII, Aniara Corporation) according to manufacturers'protocols. A standard curve was generated using serum collected fromsaline-treated animals.

Compositions with siRNA directed to Factor VIII were formulated withATX-001, ATX-002, ATX-003, and ATX-547, and comparator samples NC1 andMC3 (Alnylam). These were injected into animals at 0.3 mg/kg and at 1mg/kg. The siRNA encapsulated by MC3 (0.3 mg/kg), NC1 (0.3 mg/kg),ATX-547 (0.3 mg/kg), ATX-001 (0.3 and 1.0 mg/kg), ATX-002 (0.3 and 1.0mg/kg), and ATX-003 (0.3 and 1.0 mg/kg) was measured for the ability toknockdown Factor VII in mouse plasma following administration of thesiRNA formulation to C57BL6 mice. The results showed that ATX-001 andATX-002 were most effective at 0.3 mg/kg, compared to controls (FIGS. 1and 2).

The siRNA encapsulated MC3 (0.3 and 1.5 mg/kg), NC1 (0.3 mg/kg), ATX-547(0.1 and 0.3 mg/kg), ATX-004 (0.3), ATX-006 (0.3 and 1.0 mg/kg), ATX-010(0.3 mg/kg), and ATX-001 (0.3 and 1.5 mg/kg), was measured for FactorVII knockdown in mouse plasma following administration of the siRNAformulation to C57BL6 mice. The results showed that ATX-001 and ATX-010were most effective (FIGS. 3 and 4). The knockdown activity of theexemplary compounds is shown for 0.3 mg/kg or at 0.05 mg/kg for ATX-018,ATX-019, and ATX-020 (Table 1).

What is claimed:
 1. A compound of formula I

wherein R is a linear alkyl of 1 to 12 carbons, or a linear alkenyl oralkynyl of 2 to 12 carbons; L is a linear alkylene or alkenylene of 5 to18 carbons; X is —CO—O— or —O—CO—; Y is S or O; R₁ is a linear orbranched alkylene consisting of 1 to 6 carbons; and R₂ and R₃ are thesame or different, consisting of a hydrogen or a linear or branchedalkyl consisting of 1 to 6 carbons; and n is 1-6; or a salt or solvatethereof.
 2. The compound of claim 1, wherein X is —CO—O—.
 3. Thecompound of claim 1, wherein X is —O—CO—.
 4. The compound of claim 1,wherein Y is S.
 5. The compound of claim 1, wherein Y is O.
 6. Thecompound of claim 1, wherein n is
 1. 7. The compound of claim 1, whereinR is an alkenyl.
 8. The compound of claim 1, wherein L is an alkylene.9. The compound of claim 1, wherein L is an alkenylene.
 10. The compoundof claim 1, wherein L and R together have 14 to 20 carbons.
 11. Thecompound of claim 1, wherein L and R together have 16 carbons.
 12. Thecompound of claim 1, wherein R₁ is a C2 or a C3.
 13. The compound ofclaim 1, wherein R₂ and R₃ together have 2 to 4 carbons.
 14. Thecompound of claim 3, wherein the compound is selected from the groupconsisting of formulas i to vi


15. A composition of the compound of claim 1, further comprising a lipidparticle.
 16. A composition of the compound of claim 1, furthercomprising an RNA, wherein the RNA is encapsulated.
 17. A composition ofthe compound of claim 1, further comprising a lipid conjugate.
 18. Acomposition of the compound of claim 1, further comprising anon-cationic lipid.
 19. A compound selected from the group consisting offormulas ATX-001, ATX-004 to ATX-008, ATX-014, ATX-016, ATX-021,ATX-023, ATX-027, ATX-028, and ATX-030